A key function of the intestinal epithelium is to discriminate commensal from pathogenic microbes and thus avoid infectious disease and chronic inflammation; however, little is known about how intestinal epithelial cells (IECs) sense bacteria at the apical surface and transduce the signals to drive appropriate transcriptional responses. The long-term goal of this research is to elucidate relationships between IEC and microbe regulatory networks that determine the outcome of host-microbe interactions in vivo. The overall objective of this proposal is to elucidate pathways mediated by MITF-TFE (MiT) family transcription factors (TFs) that determine the host IEC response to infection. Because MiT family TFs are highly conserved, we chose to use Caenorhabditis elegans, a genetically tractable model organism, as a tool to address these questions. C. elegans exhibits pathogen-specific responses that are independent of TLR, NLR, and NF-kB, and thus represents a useful tool to study novel host defense pathways that are evolutionarily conserved, in an unbiased manner in vivo. The central hypothesis of this proposal is that unknown signaling pathways activated during pathogenic infection control MiT-mediated host defense in C. elegans and human IECs. The rationale for the proposed research is that, once it is understood how MiT TFs function in the host response and how they are regulated in vivo, their activity could likely be manipulated pharmacologically, resulting in new and innovative approaches for the prevention and treatment of a variety of infectious or inflammatory diseases. To test the central hypothesis, three specific aims are proposed: 1) Elucidate upstream regulators of HLH-30/MiT, using mosaic genetic analysis, biochemical characterization of post-translational modifications, and genetic analysis of candidate upstream pathways; 2) Elucidate downstream target pathways of HLH-30/MiT, using global gene expression profiling and chromatin immunoprecipitation approaches, and 3) Elucidate the role of MiT TFs in human IEC host defense, using human IEC lines in biochemical, molecular, and cell biological approaches cou- pled with high-throughput gene expression profiling. The contribution of the proposed research is expected to be the elucidation of novel, non-TLR/NLR/NF-B, HLH-30/MiT-mediated pathways regulating the host response to bacteria in C. elegans and human IECs. These contributions are significant because they are the first step in a continuum of research that will eventually allow pharmacologic manipulation of host defense via MiT signaling, either positively or negatively, to treat infection or inflammatory disease. The proposed research is conceptually innovative because it shows for the first time that MiT TFs are differentially expressed in human IECs during microbial stimulation and intestinal inflammation.Furthermore, the proposed research is innovative because it represents a departure from more directed in vitro approaches and takes advantage of the tractability of C. elegans, and thus is an unbiased and efficient in vivo approach. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the signaling components identified upon successful completion of these studies are likely to provide new targets for prevention and treatment of intestinal infectious or inflammatory disease and significantly advance the field of host-microbe interactions. The research proposed here is expected to enable further exploration of the role of MITF/TFE (MiT) transcription factors, in the context of other signaling pathways in vertebrate genetic models of intestinal infection and inflammatory disease. The project is relevant to the mission of the NIH because the knowledge acquired with the proposed studies will increase the repertoire of pathways that control host defense and MRSA pathogenesis, leading to greater understanding of disease, enabling research to identify new targets for treatment, and having the po- tential to accelerate the development of new therapies, reducing the burden of a variety of infections and in- flammatory diseases.